Antidiabetic properties of the ethanolic extract of Rhus coriaria fruits in rats

Background and the purpose of the study Fruits of Rhus coriaria L. (Anacardiaceae) are traditionally used as a table spice in Iran and are highly recommended for diabetic patients. The purpose of this study was to determine the antidiabetic properties of the ethanolic extract of Rhus coriaria fruits and also its mechanisms of action. Methods The effects of ethanolic extract of Rhus coriaria fruits were measured on blood glucose, lipids and antioxidant enzymes by commercial kits. mRNA levels of insulin (INS) and glucose transporter type-4 (GLUT-4) genes were investigated by RT-PCR (Reverse transcription- polymerase chain reaction) technique. Moreover, its effects on intestinal a-glucosidases was measured using an in vitro method. Results and Conclusion Following a single dose administration of the extract it was found that extract could significantly reduce postprandial blood glucose by 24% (at 5 hrs). In the long term experiment, on the day of 21, postprandial blood glucose (PBG) was found to be significantly lower (by 26%) compared to diabetic control group. The plant extract raised markedly serum high-density lipoprotein (HDL) by 34% and also reduced low-density lipoprotein (HDL) by 32%. Also it had noticeable antioxidant effects by elevating superoxide dismutase (SOD) and catalase(CAT) activities by 46% and 77%, respectively. However it did not show a strong effect on glutathione peroxidase (GPX) activity. The extract inhibited maltase and sucrase activities by 44% and 27%, respectively. However it made no changes in the transcript levels of INS and GLUT-4 genes. It can be concluded that constituents of Rhus coriaria fruits have effective components which can be utilized as useful herb for alleviation of diabetes complications.


INTRODUCTION
Type 2 diabetes is a major public health concern, causing tens of millions of chronic illnesses and a significant number of deaths worldwide each year (1). It is characterized by hyperglycemia and profound alterations in the plasma lipids and lipoproteins profiles (2). Also antioxidant defense system which normally modulates the level of oxidative stress (3) is altered in diabetes (4) and it involves in the pathogenesis of diabetes and its complications (5). Therefore glycemic, lipids profiles and oxidative stress control is fundamental for the management of diabetes (6). In Iran, Rhus coriaria is traditionally used as a table spice especially along with rich dishes and is highly recommended for adjustment of the blood lipids in diabetic patients. Previous studies on this plant have shown that its fruits (7) and leaves (8) have in vitro antioxidant properties. Also it has already been reported that in vitro hypoglycemic activity of the methanolic extract of fruits of Rhus coriaria is due to inhibition of α-amylase (87% inhibition at 50 μg/ml) (9). In this study the antihyperglycemic, hypolipidemic and anioxidant activities of the ethanolic extract of Rhus coriaria fruits on diabetic male Wistar rats was investigated in order to verify the use of this herbal medicine in treatment of type 2 diabetes.

MATeRIAl AND MeThODS
Chemicals and reagents DEPC Water, Taq polymerase and RNA extraction kit were purchased from CinnaGen (Tehran, Iran). DNase I, RNase free kit was from Fermentas (Ontario, Canada), RT kit and Primers were from Bioneer (Daejon, Korea), dNTPs were from BioFlux (Tokyo, Japan). The kits for determination of glucose, triglyceride (TG), total cholesterol (TC), high density lipoprotein (HDL) were from Chem Enzyme (Tehran, Iran). Hemoglobin Reagent Set was from Ziest Chem (Tehran, Iran), SOD and GPX kits were from Randox (Antrim, UK). All other chemicals and solvents were of the highest commercial grade from Merck (KGaA, Germany) or Sigma (St Louis, MO, USA).

Preparation of Rhus coriaria fruits ethanolic extract (RE)
Fruits of R. Coriaria were collected from Markazi province of Iran and were authenticated by Professor Ahmad Qahraman and a Voucher specimen (No. 5084) was deposited in the herbarium of University of Tehran (Tehran, Iran). The air dried fruits of R. coriaria were finely powdered (400 g) and extracted three times with fresh 96% ethanol at room temperature for 12 hrs. The ethanolic solution after removal of solids was concentrated at 40°C by rotary evaporator and then lyophilized. The percentage yield based on the dried starting material was 16%. The powder was stored at dark at 4°C for subsequent experiments.

Preparation of Alloxan-induced diabetic wistar rats
Male Wistar rats (Rattus norvegicus) weighing 200-250 g were used in this study (Pasteur Institute, Tehran, Iran). Animals were housed six per standard rat cage, in a room with a 12:12 hrs light/dark cycle and controlled temperature (22 ± 1 °C). Diabetes was induced in overnight fasted rats by subcutaneous injection of alloxan monohydrate (100 mg/kg, Sigma, St Louis, MO, USA), dissolved in citrate buffer (pH = 4.5), according to a previously described method (10).

Methods
The rats were divided into five groups of six each. Group I (NC): normal rats were treated with vehicle alone; group II (DC): diabetic rats were treated with vehicle alone; group III (REa+D): diabetic rats were treated with RE at doses of 200 mg/kg BW; group IV (REb+D): diabetic rats were treated with RE at doses of 400 mg/kg BW. Group V (Ac+D in the1st phase or Met+D in the 2nd phase): diabetic rats were treated with acarbose (20 mg/kgBW) or metformin (100mg/kg BW). The doses were selected from pervious publications (11). In the first phase of this study, following 1, 3, 5, 8 and 24 hrs of oral administration of a single dose of samples to rats, PBG levels were estimated using blood obtained from tail vein and a glucometer (On Call Now, San Diego, USA). After two days, Oral Glucose Tolerance Test (OGTT) was carried out for all rats. In this phase acarbose was used as the reference drug. In the second phase, two days after OGTT, all rats were administered once a day with samples for 21 days. In this phase metformin was used as the reference drug. PBG levels were estimated at the end of one, two and three weeks of treatments. At the end of treatment period, rats were anesthetized by ether and their bloods were collected. Serum total cholesterol, triglycerides and high density lipoprotein levels and the activities of SOD (EC: 1.15.1.1) and GPX (EC: 1.11.1.9) were measured using commercial kits. Low density lipoprotein and very low density lipoprotein were calculated by formula (12) and CAT (EC: 1.11.1.6) activity was measured by method of Aebi (13).Then the animals were killed and their pancreases and hearts were removed promptly for the estimation of insulin (INS) and glucose transporter-4 (GLUT-4) mRNA expression. In a separate experiment the inhibitory effects of plant extract on intestinal α-Glucosidases (sucrase and maltase) was measured by an in vitro method.
Measurement of RE effects on oral glucose tolerance NC and DC Groups received distilled water orally. Animals of REa+D and REb+D groups received orally RE, 200 and 400 mg/kg BW respectively.To group Ac+D was given the reference drug, acarbose, (20 mg/kg BW). Thirty min later, each rat received orally a carbohydrate solution (equal proportion of maltose and sucrose) (2 g/kg BW). PBG of each animal was determined at 0 min, just before carbohydrate solution loading, and at 30, 60 and 120 min after that, using a glucometer (On Call Now, San Diego, USA).

Gene expression analysis
Pancreas and heart left ventricle tissues of DC, NC and REb+D groups were dissected from rats immediately after sacrificing, placed in liquid nitrogen, and then stored at -70°C until used. Total RNA of tissues were extracted by RNX-Plus kit according to its manual. The DNA free RNA was prepared prior to RT-PCR using DNase I, RNase-free kit. For reverse transcription to obtain cDNA, AccuPower RT PreMix kit, 50 ng/μl template RNA and 25 ng/μl oligo dT18 were used. The primers were as follows: and B-act F, 5′-AGCCATGTACGTAGCCATCC-3′; B-act R, 5′-TCTCAGCTGTGGTGGTGAAG-3′ (248-bp). For PCR reaction, 500 ng of the cDNA was added to a PCR reaction mixture consisting of 10X PCR buffer (2.5 μl), 50 MmMgCl 2 (0.75 μl), 10 mM dNTPs (0.5 μl), 10 pM of paired primers (0.5 μl of each), 0.25 units of Taq polymerase and distilled water in a total volume of 25 μl. The reaction mixture was overlaid in a PCR thermal cycler for 35 cyclic reactions. PCR products were run on 1.5% agarose gels, stained with ethidium bromide and photographed. Images of radiographs were analyzed with TotalLab v1.10 using 1D analysis.

In vitro inhibition assay for rat intestinal sucrase and maltase activities
The inhibitory effects of RE on rat intestinal sucrase and maltase activities were determined using a literature method (14).

Statistical analyses
All data are presented as means±S.D. for six rats in each group. Comparison between groups and between time points was made by one-way analysis of variance (ANOVA) followed by Duncan's test using SPSS (SPSS Inc, Chicago, USA).

Short-term effects of RE on PBG levels
Administration of the extract at the dose of 400 mg/kg reduced the glucose level at 5 hrs time point compared to both initial level and DC group ( Table 1).

Effects of RE on oral glucose tolerance
As shwon in table 2, diabetic rats treated with 200 and 400 mg/kg RE showed remarkable decrease in blood glucose at the first hour after carbohydrate solution loading compared to DC group;however they were not as effective as acarbose.

Long-term effects of RE on PBG
The RE at the dose of 400 mg/kg induced significant reductions in PBG levels in diabetic rats, after 7, 14 and 21 days of treatment (Table 3), compared to initial time point. The data of REb+D and Met+D groups were significantly different from those of DC group (p<0.0001).

Effects of RE on rat intestinal sucrase and maltase activities
The crude ethanolic extract of Rhus coriaria fruits showed strong inhibitory effects for both sucrase and maltase activities,25.38%( ± 2.05) and 44.33%(±1.14) respectively.

Effects of RE on INS and GLUT-4 genes expression
Densitometric scanning revealed no increase in INS (p>0.156) and GLUT-4 (p>0.564) transcripts by RE, as compared to DC group.

Effects of RE on blood lipids
Treatment with RE (400 mg/kg) strongly increased (  coriaria fruits increased SOD and CAT levels of the red blood cells. This finding upholds the in vitro antioxidant activity of Rhus coriaria demonstrated perviously (7). Antioxidant activity of Rhus coriaria could also be useful for prevention of diabetes complications due to hyperglycemia (11). This study aside from Rhus coriaria antihyperglycemic and antioxidant activities indicated that extract may also reverse dyslipidemia associated with diabetes by increasing the HDL level and decreasing the LDL level similar to some other herbs previously described (18).
In conclusion, the use of Rhus coriarisa in Iranian folk medicine as a hypolipidemic agent has a significant correlation with the scientific data generated in this study. Also the present study explains two other properties of Rhus coriaria fruits on diabetic rats which can improve the life of type 2 diabetic patients: mild antihyperglycemic and potent antioxidant peroperties. Further studies are required to show the active constituent(s) of Rhus coriaria.